Anti-sulfadiazine nanoantibody and its application

Abstract

The present invention discloses an anti-sulfadiazine nano-antibody and its application, belonging to the field of nano-antibody technology. In order to provide a nano-antibody for detecting sulfadiazine that has a small molecular weight, strong penetrating power, good stability, strong antigen binding ability, and can recognize antigen epitopes that are difficult for conventional antibodies to recognize. The present invention provides a nano-antibody for detecting sulfadiazine, the amino acid sequence of CDR1 of the variable region of the nano-antibody is shown as SEQ ID NO.6, the amino acid sequence of CDR2 of the variable region of the nano-antibody is shown as SEQ ID NO.7, and the amino acid sequence of CDR3 of the variable region of the nano-antibody is shown as SEQ ID NO.8. The indirect competitive ELISA method established by the present invention is a simple, rapid, sensitive, and low-cost immunoassay method, which has great application prospects in the detection of sulfadiazine residues in animal-derived foods.

Description

Technical Field

[0001] The present invention belongs to the field of nano antibodies, and particularly relates to an anti-sulfadiazine nano antibody and applications thereof. Background Art

[0002] Sulfadiazine (SD) is a common anti-systemic infection drug with a molecular formula of C 10 H 10 N₄O₂S, with a molecular weight of 250.28 Da, appears as white or off-white crystals or powder that gradually darken in light. It is slightly soluble in ethanol and acetone and virtually insoluble in water. Its basic structure is similar to para-aminobenzoic acid (PABA). It can compete with PABA for the dihydrofolate synthase enzyme in bacteria, affecting folate metabolism and nucleotide synthesis, thereby inhibiting bacterial growth and reproduction. SD, with its low toxicity, high potency, broad antimicrobial spectrum, and readily absorbed properties, is widely used to treat bacterial infections in aquaculture, medicine, and animal husbandry. With a half-life of over 10 hours, SD belongs to the intermediate-acting sulfadiazine class. Due to its irregular use and even abuse, SD residues are common in various animal-derived foods, compromising food safety and posing a serious threat to human health. Therefore, strengthening the detection of SD residues is crucial.

[0003] Currently, the main analytical methods commonly used to detect sulfonamide residues include high-performance liquid chromatography, liquid chromatography-mass spectrometry, and immunoassays. While chromatographic methods offer high sensitivity and good accuracy, they require stringent sample pretreatment procedures, requiring standardized pretreatment steps for testing. Furthermore, the specialized instruments used are often large and expensive, requiring specialized personnel training and strict test procedures. The limited number of sample injections available from the equipment makes them inadequate for large-scale monitoring of drug residues in animal-derived foods. Among immunoassays, the enzyme-linked immunosorbent assay (ELISA) is the most common. Its simplicity and low cost make it ideal for large-scale, field-based drug residue surveys. Therefore, developing cost-effective and efficient methods for detecting sulfonamide residues is of practical significance. In immunoassay research, indirect competitive ELISA has been widely used in veterinary drug residue testing due to its high efficiency and convenience. However, most methods utilize traditional polyclonal or monoclonal antibodies as detection antibodies, which present technical challenges such as poor stability, weak antigen-binding capacity, and difficulty recognizing antigenic epitopes. Summary of the Invention

[0004] The purpose of the present invention is to provide a nano-antibody for detecting sulfadiazine, which has small molecular weight, strong penetrating power, good stability, strong antigen binding ability and can recognize antigen epitopes that are difficult for conventional antibodies to recognize.

[0005] The present invention provides a nanobody for detecting sulfadiazine, wherein the amino acid sequence of CDR1 of the variable region of the nanobody is shown as SEQ ID NO.6, the amino acid sequence of CDR2 of the variable region of the nanobody is shown as SEQ ID NO.7, and the amino acid sequence of CDR3 of the variable region of the nanobody is shown as SEQ ID NO.8.

[0006] It is further defined that the Nanobody also includes four framework regions, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.5.

[0007] The present invention provides a nucleotide encoding the above-mentioned Nanobody.

[0008] The present invention provides a nano antibody for detecting sulfadiazine, and the amino acid sequence of the nano antibody is shown in SEQ ID NO.1.

[0009] The present invention provides a recombinant vector containing the above-mentioned nucleotide.

[0010] The present invention provides a recombinant microbial cell containing the above-mentioned nucleotide.

[0011] The present invention provides a kit for detecting sulfadiazine, wherein the kit contains the nano antibody.

[0012] The present invention provides a use of the nanobody, the nucleotide sequence, the recombinant vector or the recombinant microbial cell in preparing a kit for detecting sulfadiazine.

[0013] The present invention is further defined as detecting sulfadiazine residues in the food field, the environmental field or the medical field.

[0014] The present invention provides a method for detecting sulfadiazine residues, which utilizes the above-mentioned nanoantibody to perform indirect competitive ELISA.

[0015] Beneficial effects: The present invention uses the synthetic complete antigen SD-BSA to screen the amino acid sequence of the anti-SD specific nanoantibody Nb1-B3 from the alpaca natural nanolibrary, recombines it with the pET28a prokaryotic expression vector, and obtains the nanoantibody Nb1-B3 with higher purity after induction purification.

[0016] The indirect competitive ELISA method based on Nb1-B3 had an IC50 value of 26.08 ng / mL, a linear detection range of 3.57 to 169.88 ng / mL, a limit of detection of 2.85 ng / mL, and a limit of quantification of 3.46 ng / mL. This method demonstrated high specificity, accuracy, and precision.

[0017] The indirect competitive ELISA method established in the present invention is a simple, rapid, sensitive and low-cost immunoassay method and has great application prospects in the detection of sulfadiazine residues in animal-derived foods.

[0018] The thermal stability analysis of the prepared nanobody Nb1-B3 showed that it retained 90% of its activity after heating at 95°C for 5 minutes, and 80% of its binding activity after heating at 80°C for 1 hour. This shows that the nanobody has high thermal stability.

[0019] The prepared Nanobody Nb1-B3 was subjected to an organic solvent tolerance analysis. Nb1-B3 retained approximately 40% activity in 40% methanol and 60% acetonitrile, while approximately 30% activity was retained in 40% acetone and 60% DMSO. This demonstrates that the Nanobody has good organic solvent tolerance.

[0020] Compared with monoclonal antibodies (150-200kDa), nanobodies (about 15kDa) have a smaller molecular weight, thus exhibiting higher penetration. Due to their small size, nanobodies can bind to antigenic epitopes that are difficult for traditional antibodies to recognize, exhibiting higher affinity activity. At the same time, nanobodies can still maintain binding to target antigens under high temperatures, organic solvents and different pH environments. The industrial large-scale production process of monoclonal antibodies is complex and costly. In contrast, nanobodies can be efficiently expressed in prokaryotic expression systems, with higher yields and lower production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the molecular structural formula of sulfadiazine;

[0022] Figure 2 Synthesis route for SD-BSA and SD-OVA;

[0023] Figure 3 UV scanning images of SD-BSA and SD-OVA; A is SD-BSA protein, B is SD-OVA protein;

[0024] Figure 4The figure shows the identification of SD-BSA and SD-OVA by gel electrophoresis; M is protein marker, 1 is BSA, 2 is SD-BSA, 3 is OVA, and 4 is SD-OVA;

[0025] Figure 5 This is the result of Phage ELISA identification;

[0026] Figure 6 is the PCR identification result of positive clones; M is DNA molecular marker DL2000, 1 is ER2738 negative control, and 2 is 1-B3;

[0027] Figure 7 The double enzyme digestion verification of Nb-pET28a; M is the DNA molecular marker DL5000, 1 is pET28a, 2 is Nb1-B3-pET28a, and 3 is Nb1-B3-pET28a after enzyme digestion;

[0028] Figure 8 Western-blotting and SDS-PAGE analysis of nanobody Nb1-B3; A is Western-blotting analysis, B is SDS-PAGE analysis;

[0029] Figure 9 For the analysis of the antigen binding activity of the nanobody Nb1-B3;

[0030] Figure 10 Thermal stability analysis of the nanobody Nb1-B3; A represents Nb1-B3 incubated at 20-95°C for 5 min, and B represents Nb1-B3 incubated at 80°C for 10-60 min.

[0031] Figure 11 This is the organic reagent tolerance analysis of nanobody Nb1-B3; A is methanol, B is acetonitrile, C is acetone, and D is dimethyl sulfoxide;

[0032] Figure 12 This is the standard curve for the detection of sulfadiazine by indirect competitive ELISA based on Nb1-B3. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0034] Example 1. Synthesis and identification of sulfadiazine complete antigen

[0035] The chemical formula of sulfadiazine described in the following examples of the present invention is as follows Figure 1 shown.

[0036] 1. Synthesis of SD-BSA and SD-OVA artificial antigens

[0037] The present invention studies the synthesis of complete antigens SD-BSA and SD-OVA by diazotization. The synthesis route is shown in Figure 2 The specific operation method is as follows:

[0038] Weigh 10 mg of SD and dissolve it in 5 mL of 0.2 mol / L HCl. After cooling in an ice bath, add 1 mol / L NaNO2 dropwise until the starch potassium iodide test paper turns blue-black. Stir and react at 4°C for 6 hours. Then, slowly add 2 mL of 10 mg / mL bovine serum albumin (BSA) solution dropwise, adjust the pH to 8.5, and continue the reaction at 4°C overnight. The reaction product is dialyzed against PBS at 4°C for 3 days, with the solution changed three times daily to remove unreacted small molecules. The complete antigen SD-BSA is obtained. The coating antigen SD-OVA is prepared using the same method and stored in small aliquots at -20°C.

[0039] 2. UV scanning identification

[0040] To identify whether the small molecule drug is successfully coupled to the protein, the SD, BSA, OVA, SD-BSA, and SD-OVA protein solutions were scanned at all UV wavelengths using a UV spectrophotometer, and their UV absorption spectra were measured to analyze changes in characteristic absorption peaks. Figure 3 As shown, the UV scanning identification spectrum shows that SD has an absorption peak at 256nm, BSA and OVA have protein-specific absorption peaks at 278nm and 280nm, respectively, while the absorption peaks of SD-BSA and SD-OVA are shifted, which indicates that the UV characteristic absorption peaks of BSA and OVA change after binding to the drug. It can be inferred that after the carrier protein binds to the drug, the optical properties of the produced substance change, indicating that the coupling effect is good.

[0041] 3. SDS-PAGE identification

[0042] BSA, OVA, SD-BSA and SD-OVA before and after coupling were analyzed and identified by SDS-PAGE. Figure 4 It can be seen that the sizes of BSA and OVA bands are around 66kDa and 45kDa, which are consistent with the theoretical values. In comparison, the electrophoresis bands of artificial synthetic antigens SD-BSA and SD-OVA are slightly higher and the molecular size is slightly increased, indicating that SD is successfully coupled with BSA and OVA.

[0043] Example 2. Screening and identification of sulfadiazine-specific nanobodies

[0044] 1. Screening of sulfadiazine nanoantibodies

[0045] Take 300 μL of natural nanoantibody library and add it to 100 mL of 2×YT (ATG) liquid culture medium, and culture at 37°C until OD 600 The cell culture medium was 0.6. Helper phage M13KO7 was added at an MOI of 20:1 and the cells were gently shaken in a 37°C water bath for 30 minutes, followed by shaking for 30 minutes. The bacterial suspension was centrifuged at 3300 × g for 10 minutes at 4°C. The pellet was resuspended in 100 mL of 2×YT (ATK) and incubated with shaking at 30°C overnight. Two immunotubes were coated with 100 μg / mL SD-BSA and BSA, respectively, overnight at 4°C, with a coating volume of 2 mL. The next day, the bacterial suspension was centrifuged at 10,000 × g for 10 minutes at 4°C. The supernatant was aspirated and added to 1 / 4 volume of ice-cold PEG / NaCl. The supernatant was then ice-cold for 50 minutes and centrifuged again at 10,000 × g for 20 minutes at 4°C. The supernatant was removed, the pellet was resuspended in 2.4 mL of PBS, and centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatant was the phage-recombinant antibody library. 100 μL of phage supernatant was serially diluted and added to the ER2738 host bacteria. The cells were gently shaken in a 37°C waterbath for 30 minutes. The cells were then plated onto SOB (GTK) plates and incubated overnight at 30°C to calculate the input volume for one round of the recombinant antibody library. The recombinant antibody library was then transferred to an immunotube that had been pre-coated with BSA overnight. The reaction was incubated at room temperature for 30 minutes. 1.5 mL of the liquid from the immunotube was then transferred to a 5 mL EP tube and 1 mL of 3% MPBS was added for 20 minutes to eliminate interference. 2 mL of the treated recombinant antibody library was then transferred to an SD-BSA-coated immunotube. The tube was gently shaken at 37°C for 30 minutes and then allowed to stand for 90 minutes. The supernatant was discarded and the cells were washed three times with PBST and then three times with PBS. After patting dry, 2 mL of 0.1 mol / L triethylamine solution was added. The cells were gently shaken at 37°C for 8 minutes for elution. An equal amount of Tris-HCl was immediately added to neutralize the mixture. This was the eluted phage. The eluted phage antibody library was added to ER2738 host bacteria in the logarithmic growth phase and infected with gentle shaking in a 37°C water bath for 30 minutes. 100 μL of the dilution was spread onto SOB (ATG) plates to calculate the output of one round. The remaining bacterial solution was centrifuged at 3300 × g for 10 minutes to resuspend the pellet, spread onto SOB (ATG) square culture plates after dilution, and the colonies were scraped the next day to obtain the primary recombinant antibody library.

[0046] Four rounds of panning were performed using the same method, with SD-BSA protein coating concentrations of 100 μg / mL, 75 μg / mL, 50 μg / mL, and 10 μg / mL, respectively. The number of PBST and PBS washes was increased by three times per round, resulting in a quaternary recombinant antibody library.

[0047] Table 1 Four rounds of panning enrichment results

[0048] First round Second round Round 3 Round 4 enter <![CDATA[1.2×10 11 ]]> <![CDATA[1.3×10 11 ]]> <![CDATA[9.8×10 9 ]]> <![CDATA[1.2×10 10 ]]> Output <![CDATA[8.0×10 3 ]]> <![CDATA[1.4×10 4 ]]> <![CDATA[5.6×10 5 ]]> <![CDATA[3.8×10 5 ]]> Enrichment rate <![CDATA[6.7×10 -8 ]]> <![CDATA[1.1×10 -7 ]]> <![CDATA[5.7×10 -5 ]]> <![CDATA[3.1×10 -5 ]]>

[0049] Note: Enrichment rate = phage output (pfu) / phage input (pfu)

[0050] 2. Phage ELISA identification

[0051] 48 monoclonal clones obtained from the fourth round of enrichment were used to prepare phage supernatants. The binding activity of the recombinant phage to SD antigen was detected by ELISA. The clones with an OD value ratio of greater than 2.0 between the experimental group coated with SD-OVA and the negative control group coated with OVA were considered positive. Figure 5 shown.

[0052] 3. PCR identification and sequencing analysis

[0053] The positive clone strain 1-B3 was selected to amplify the VHH gene, and the results of colony PCR identification were as follows: Figure 6 As shown, compared with the negative control, a band of about 450bp was amplified, indicating that the VHH gene of the selected clone strain was successfully inserted. The positive clone strain 1-B3 was sequenced and analyzed, and the amino acid sequence was shown in SEQ ID No.1, which has four complete FR framework regions and three CDR variable regions. The amino acid sequence of FR1 is shown in SEQ ID No.2, the amino acid sequence of FR2 is shown in SEQ ID No.3, the amino acid sequence of FR3 is shown in SEQ ID No.4, and the amino acid sequence of FR4 is shown in SEQ ID No.5; the amino acid sequence of CDR1 is shown in SEQ ID No.6, the amino acid sequence of CDR2 is shown in SEQ ID No.7, and the amino acid sequence of CDR3 is shown in SEQ ID No.8.

[0054] Example 3. Preparation of anti-sulfadiazine nanobody

[0055] 1. Construction of prokaryotic expression recombinant plasmid

[0056] The positive single clone was extracted from the phagemid for sequencing. After the sequencing was correct, it was used to construct a prokaryotic expression recombinant plasmid according to the sequence of the expression strain. BamH I and Xho I restriction sites were added to the 5' end and 3' end respectively, and the pET28a recombinant expression plasmid was synthesized by Heilongjiang Jiansu Gene Technology Co., Ltd. The recombinant plasmid (in DH5α) was coated on an LB solid plate and inverted and cultured at 37°C overnight. The next day, a single clone was picked, the extracted plasmid was identified by double enzyme digestion, and then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Correct sequencing indicated that the prokaryotic expression vector was successfully constructed, and the extracted plasmid was transformed into BL21 (DE3) competent medium for prokaryotic expression. The results after double enzyme digestion with BamH I and Xho I are shown in Figure 7Compared with the empty plasmid and the constructed prokaryotic expression vector, an inserted target fragment of about 450bp appeared, proving that the prokaryotic expression recombinant plasmid was successfully constructed.

[0057] 2. Soluble expression and purification of nanobodies

[0058] After the glycerol strains were revived at 37°C, they were transferred to a large bottle of 500 mL LB-K liquid medium at a ratio of 1:100 and continued to shake culture until the OD 600 After reaching the logarithmic phase value, the bacterial solution was cooled in an ice bath and allowed to stand, and then IPTG inducer with a final concentration of 0.8mM was added and induced at 18℃ for 16h. The bacterial precipitate was collected the next day and weighed. 30mL of bacterial active protein extraction reagent was added per gram of wet bacteria. At the same time, 2mg / mL of lysozyme and 100mM PMSF protease inhibitor were added to ensure the protein extraction efficiency. The protein was purified by nickel column affinity chromatography, and the eluate was then taken for Western blotting and SDS-PAGE verification. The results are as follows Figure 8 As shown in the figure, a single protein band appeared at about 15 kDa, indicating that the purified protein had a high purity.

[0059] The amino acid sequence of the obtained nanobody with specific anti-SD is shown in SEQ ID NO.1.

[0060] The antibody sequence includes four framework regions (FR1, FR2, FR3, FR4) and three complementary determining regions (CDR1, CDR2, CDR3). The arrangement order of the four framework regions and the three complementary determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0061] The amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.5; the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.8.

[0062] SEQ ID NO.1:

[0063] QVQLQESGGGLVQPGGSLTLSCVVSGSIFSSDAVVWYRQAPGSERELVAAMRSGGHT YYPDSVKDRFTISRDYDKNTIYLQMTSLKPDDTAMYYCAASRPGLKYYSDGYYSQSDFNL WGQGTQVTVSS;

[0064] SD nanobody gene sequence, SEQ ID NO.9:

[0065] caggtgcagctgcaggagtctgggggaggcttggtgcaacctggggggtctctgacactctcctgtgtagtctctggaagcatcttcagtagcgatgccgtggtctggtatcgtcaggctccagggagcgagcgcgagttggtcgcggctatgcgaagcggaggccacacatactaccctgattctgtgaaggaccgattcaccatctccagagactatgacaagaatacaatttatttgcaaatgaccagcctgaaacctgacgacacggccatgtattactgtgcggcatcccggcccggactgaaatactatagtgatggttactactcccaatctgactttaatttatggggccaggggacccaggtcaccgtctcctcac;

[0066] SEQ ID NO.2: QVQLQESGGGLVQPGGSLTLSCVVS;

[0067] FR1 gene sequence, SEQ ID NO.10:

[0068] caggtgcagctgcaggagtctgggggaggcttggtgcaacctggggggtctctgacactctcctgtgtagtctct;

[0069] SEQ ID NO.3: VVWYRQAPGSERELVAA;

[0070] FR2 gene sequence, SEQ ID NO.11:

[0071] gtggtctggtatcgtcaggctccagggagcgagcgcgagttggtcgcggct;

[0072] SEQ ID NO.4: YYPDSVKDRFTISRDYDKNTIYLQMTSLKPDDTAMYYC;

[0073] FR3 gene sequence, SEQ ID NO.12:

[0074] tactaccctgattctgtgaaggaccgattcaccatctccagagactatgacaagaatacaatttatttgcaaatgaccagcctgaaacctg acgacacggccatgtattactgt;

[0075] SEQ ID NO.5:WGQGTQVTVSS;

[0076] FR4 gene sequence, SEQ ID NO.13:

[0077] tggggccaggggacccaggtcaccgtctcctcac;

[0078] SEQ ID NO.6: GSIFSSDA;

[0079] CDR1 gene sequence, SEQ ID NO.14:

[0080] ggaagcatcttcagtagcgatgcc;

[0081] SEQ ID NO.7: MRSGGHT;

[0082] CDR2 gene sequence, SEQ ID NO.15:

[0083] atgcgaagcggaggccacaca;

[0084] SEQ ID NO.8:AASRPGLKYYSDGYYSQSDFNL.

[0085] CDR3 gene sequence, SEQ ID NO.16:

[0086] gcggcatcccggcccggactgaaatactatagtgatggttatactcccaatctgactttaattta.

[0087] Example 4. Biological properties of anti-sulfadiazine nanobody Nb1-B3

[0088] 1. Antigen binding ability analysis

[0089] Nb1-B3 was diluted to different gradients and added to the enzyme-labeled plate containing 5 μg / mL of the coating original SD-OVA. The OD value of the binding of different concentrations of nanoantibodies to the coating original was evaluated by ELISA. 450 The binding activity with target antigen is shown in Figure 9 , indicating that it has good ability to bind to antigens.

[0090] 2. Thermal stability analysis

[0091] The nanobody was diluted to a working concentration (5 μg / mL) and heated in metal baths at 20°C, 30°C, 45°C, 60°C, 80°C and 95°C for 5 minutes. Another portion was placed at 80°C for 10 to 60 minutes, and samples were taken every 10 minutes. After the antibody returned to room temperature, the ELISA method was used to evaluate the binding efficiency of the antibody to the antigen. The binding efficiency of the untreated nanobody to the antigen was 100%, and the temperature tolerance of the nanobody was evaluated. The results are shown in Figure 10 In Figure A, the activity of the nanoantibody remains stable as the heating temperature increases. After heating at 95°C for 5 minutes, it still has 90% activity. The binding activity of Nb1-B3 after incubation at 80°C for different times is shown in Figure 4. Figure 10 As shown in Figure B, the nanobody still has 80% binding activity after being heated at 80°C for 1 hour. This shows that the nanobody has high thermal stability.

[0092] 3. Analysis of organic solvent tolerance

[0093] Different concentrations (10%, 20%, 40% and 60%) of organic solvents (methanol, acetonitrile, acetone and dimethyl sulfoxide) were used for dilution to adjust the nanobody to an appropriate working concentration. The binding efficiency of the nanobody diluted with PBS to the antigen was 100%. The tolerance of the nanobody in different organic solvents and different concentrations of the same organic solvent was evaluated. Figure 11 Nb1-B3 still has about 40% activity in 40% methanol and 60% acetonitrile, and retains about 30% activity in 40% acetone and 60% DMSO. This shows that the nanobody has good tolerance to organic solvents.

[0094] Example 5. Establishment of indirect competitive ELISA method for anti-sulfadiazine nanobody

[0095] Under the optimal working conditions, the logarithm of the SD standard with a series of concentration gradients is used as the horizontal axis, and the corresponding OD 450 The ratio of the reading value to the control well is the vertical axis, and the Logistic function is used for fitting to construct the standard curve of the anti-SD nanobody. Figure 12 Indirect competitive ELISA method based on Nb1-B3 IC 50 The value was 26.08 ng / mL, the linear detection range was 3.57~169.88 ng / mL, the detection limit was 2.85 ng / mL, the quantification limit was 3.46 ng / mL, and the correlation coefficient was 0.993.

[0096] Example 6. Evaluation of the indirect competitive ELISA method of anti-sulfadiazine nanobody

[0097] 1. Specificity analysis

[0098] Six common antibiotics were selected, including three commonly used sulfonamides: sulfadiazine (SD), sulfamethazine (SM2), and sulfamethoxazole (SMZ) and three other common antibiotics: ampicillin (Amp), kanamycin (Kan), and enrofloxacin (ENR). An indirect competitive ELISA was used to draw a standard curve with the logarithm of drug concentration as the horizontal axis and the ratio of inhibition wells to control wells as the vertical axis. The IC′ of each well was calculated. 50 The calculation formula is: Cross-reactivity (CR) = IC 50 (SD) / IC′ 50 (other drugs) × 100%. The results are shown in Table 2. The cross-reactivity rates for the three non-sulfonamide drugs were all less than 0.1%, indicating essentially no cross-reactivity. The cross-reactivity rates for the three sulfonamide drugs were 14.7%, 1.2%, and 7.1%, respectively. This indicates that the detection method has low cross-reactivity only for sulfonamide drugs with a sulfonamide core, and also demonstrates that the screened nanobodies have good specificity.

[0099] Table 2 Cross-reaction rate determination

[0100]

[0101]

[0102] 2. Recovery determination

[0103] SPF porcine urine and SPF eggs were used as matrices. Porcine urine was diluted 20-fold with PBS and then spiked. Five grams of egg was homogenized and added to 20 mL of PBS. The mixture was vortexed for 1 minute and shaken for 10 minutes. A 500 μL sample of the supernatant was collected and thoroughly mixed with an equal volume of PBS buffer. The mixture was centrifuged at 5000 × g for 15 minutes. The supernatant was then spiked with SD. The drug concentrations in the spiked samples were 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. Three replicates were performed in each group. The recovery rates of the established indirect competitive ELISA method were calculated using the formula: recovery = measured value / true value × 100%. The results are shown in Table 3. Drug recoveries using porcine urine ranged from 90.29% to 96.38%, while those using egg were from 84.08% to 93.60%. The egg matrix is ​​more complex than that of porcine urine, resulting in lower recoveries compared to porcine urine.

[0104] 3. Precision determination

[0105] Precision is often measured using standard deviation or relative standard deviation, also known as the coefficient of variation (CV). The calculation formula is: CV (%) = standard deviation / mean value × 100%. According to the national guidelines for enzyme-linked immunosorbent assay kits, precision is evaluated using the coefficient of variation of the assay results, which should be no greater than 20%. The results are shown in Table 3. The coefficient of variation for porcine urine was 8.64% to 10.35%, and for egg was 10.37% to 11.41%. Both coefficients of variation were less than 12%, indicating that the established assay has good precision.

[0106] Table 3 Spike recovery determination

[0107]

Claims

1. A nanobody for detecting sulfadiazine, characterized in that The amino acid sequence of CDR1 of the variable region of the Nanobody is shown in SEQ ID NO.6, the amino acid sequence of CDR2 of the variable region of the Nanobody is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 of the variable region of the Nanobody is shown in SEQ ID NO.

8.

2. The Nanobody according to claim 1, characterized in that The nanobody also includes four framework regions, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.

5.

3. A polynucleotide encoding the Nanobody according to claim 1.

4. A nanobody for detecting sulfadiazine, characterized in that The amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

5. A recombinant vector, characterized in that The recombinant vector contains the polynucleotide according to claim 3.

6. A recombinant microbial cell, characterized in that The recombinant microbial cell contains the polynucleotide according to claim 3.

7. A kit for detecting sulfadiazine, characterized in that: The kit contains the Nanobody according to claim 1, 2 or 4.

8. Use of the Nanobody according to claim 1, 2 or 4, the polynucleotide according to claim 3, the recombinant vector according to claim 5 or the recombinant microbial cell according to claim 6 in the preparation of a kit for detecting sulfadiazine.

9. The use according to claim 8, characterized in that Detection of sulfadiazine residues in food, environmental or medical fields.

10. A method for detecting sulfadiazine residues, characterized in that: A method for performing indirect competitive ELISA using the Nanobody according to claim 1, 2 or 4.

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